DNA sequencing is considered to be among the top priorities in the biosciences field today. Advances in the sequencing of entire genomes, their annotation and variations have been the critical enabling factors for biological research. Following the completion of the human genome project, there has been continuing work in the detection of genetic variations in a large number of samples representing a broad range of biological material, which provides an insight into genetic mechanisms of different diseases.
Enzymatic chain-termination or the Sanger method, and the chemical cleavage technique of Maxam and Gilbert are examples of two methods for DNA sequencing, which rely on gel electrophoresis to resolve DNA fragments produced from a larger DNA segment.
Pyrosequencing is another method for sequencing DNA. This method does not rely on electrophoretic separation. Pyrosequencing refers to a bioluminometric DNA sequencing technique that measures the release of pyrophosphate during DNA synthesis. The existing pyrosequencing methods are typically real-time bioluminescence-based sequencing-by-synthesis methods catalyzed by four kinetically well-balanced enzymes. Such enzymes may be DNA polymerase, ATP sulfurylase, firefly luciferase and apyrase. A DNA sample, at picomole level, is usually used in the existing pyrosequencing methods based on a luciferase assay coupled with an APS-ATP surfurylase reaction for producing ATP from PPi. In the presence of DNA polymerase, correct nucleotide incorporation into the DNA sample leads to generation of pyrophosphate (PPi) in a quantity equimolar to the number of incorporated nucleotides. The released PPi then triggers the ATP sulfurylase reaction resulting in a quantitative conversion of PPi to ATP. ATP is subsequently consumed by luciferase for producing bioluminescence, which is proportional to the amount of DNA and the number of incorporated nucleotides. In pyrosequencing, the unincorporated nucleotides and the generated ATP can be degraded by apyrase allowing iterative addition of the next nucleotide dispensation to the solution. An existing pyrosequencing method based on ATP-sulfurylase may be represented by the following reaction cascade:

Even though the existing pyrosequencing methods are based on a sensitive bioluminometric assay, they still require DNA templates or samples at a picomole level, which may be challenging in some applications. Although the luminescence intensity could be increased by increasing the amount of luciferase, a large background signal due to a side reaction of APS with luciferase introduces limitations to the existing assays.
Several sequencing platforms are currently in use, however their cost can be prohibitive. Despite strong research initiatives having been launched in the area of sequencing, it appears that, at present, no technology can address the issues of cost, time and accuracy simultaneously, and in particular, to be able to sequence a human genome under about one thousand dollars USD and within a day. Therefore there remains a need for an improvement in the methods and systems for analyzing nucleic acid molecules.